The worldwide practical use of genetic recombinants, which are produced by using genetic recombinant techniques, is in progress. As the genetic recombinants which have been already practically used, genetic recombinant microorganisms such as those represented by interferon producing bacteria and genetic recombinant crops such as those represented by insect-resistant maize have been known. For example, an insect-resistant gene such as the CryIA(b) protein coding region (herein after, cryIA(b)) from Bacillus thuringiensis or herbicide-resistant gene such as phosphinotricin acetyl transferase(PAT) protein coding region (hereinafter, pat) has been used in the previously developed genetic recombinant crops. In such cases, these transgenes were introduced as the expression units combined with various DNA sequences such that the transgenes can express in the crops.
The DNA sequences which can be used include 35S promoter from cauliflower mosaic virus (hereinafter, CaMV35S promoter), the promoter of phosphoenolpyruvate carboxykinase (PEPC) gene from maize, the promoter of calcium dependent protein kinase (CDPK) from maize, the introns such as the region containing the sixth intron of alcohol dehydrogenase 1S gene from maize (Adh1-S IVS6), the region containing the second intron of alcohol dehydrogenase 1S gene from maize (Adh1-S IVS2), the 9th intron of PEPC (PEPC#9) or the intron region of heat-shock protein 70 (hsp70) from maize, and the terminators such as nopaline synthase terminator (herein after, NOS terminator) from Agrobacterium tumefaciens or 35S terminator from cauliflower mosaic virus. The actually commercially distributed genetic recombinant crops include, for example, the progeny varieties from Bt11 line of Novartis, the progeny varieties from Event 176 of Novartis, the progeny varieties from MON810 line of Monsanto, the progeny varieties from GA21 line of Monsanto and the progeny varieties from T25 line of Aventis, for maize. For soybean, they include, for example, progeny varieties from Roundup Ready Soy line. The constructions of DNAs that had been introduced into these varieties are shown in FIG. 1.
Generally, a genetic recombinant has been developed to impart an industrially preferable property to an original organism compared with the original organism. The major approach for such purpose is isolating a gene expressing the property from an organism which intrinsically exhibits the property, and introducing the gene into the interested organism such that the gene can be expressed in the organism. Thus, the DNAs from genetic recombinants include such recombinant DNA sequences which have been introduced into the recombinants.
For example, genetic recombinant crops have been developed to impart them preferable properties as agricultural products, such as insect-resistance as well as herbicide-resistance and the like, and which crops are produced by introducing the gene responsible for insect-resistance or herbicide-resistance and the like into the original crops, in the form where the gene can be expressed in the crops. Thus, the DNAs from genetic recombinants include such recombinant DNA sequences that have been introduced into the recombinants.
However, when such crops are actually commercialized, it is likely that the crops are the progeny hybrids of the genetic recombinant crops. Therefore, it should be confirmed that the introduced DNA sequences stably exist in such crops.
Additionally, European Community (EU) has enacted the regulation affecting the labeling of genetic recombinants and processed foods produced from them (Regulation (EC) No. EC/258/97, Council Regulation (EC) No. 1139/98), and the regulation affecting the labeling of genetic recombinants and processed foods produced from them was also issued in Japan, which arose the need in the food industry and its relating filed for the information about the existence and the content of recombinants in crops or in foods.
Thus, there is a need for a technique which allows the determination of the existence of genetic recombinants or the content thereof in foods, feeds and in the source crops thereof, particularly the content ratio thereof in the raw materials. When plural genetic recombinant lines are expected to be contained, a techniques is desired which makes it possible to detect them individually and to define the content ratio of each line, that is, the development of a practical technique having high sensitivity and quantitativity to detect the individual line of genetic recombinants is desired.
Although there are many reports showing that it is advantageous to use the molecular biological techniques using polymerase chain reaction (PCR) (Science Vol. 230, 1350-1354 (1985), Science Vol. 239, 487-491 (1988)) for detecting genetic recombinants, these techniques qualitatively determine whether the genetic recombinants exist or not and they do not generate the information about the content ratio of the genetic recombinants contained in the samples (see, for example, Z. Lebensm Unters Forsch A., Vol.203, 339-344 (1996), Mitt. Gebiete Lebensm. Hyg., Vol.87, 307-367 (1996), Deutsche Lebensmittel-Rundschau, Vol.93, Jahrg., Heft 2, 35-38 (1997), Z. Lebensm Unters Forsch A., Vol.205, 442-445 (1997), Zeitschrift fur Ernahrungwissenscaft Vol.36, 155-160 (1997), BGVV Hefte 1, 115-117 (1997), Mitt. Gebiete Lebensm. Hyg., Vol.88, 164-175 (1997), Mitt. Gebiete Lebensm. Hyg., Vol.88, 515-524 (1997), Food Additives and Contaminants, Vol.15, No.7, 767-774 (1998), Lebensm.—Wiss. u.—Technol., Vol.31, 664-667 (1998), Z. Lebensm Unters Forsch A., Vol.206, 203-207 (1998), Z. Lebensm Unters Forsch A., Vol.206, 237-242 (1998), Z. Lebensm Unters Forsch A., Vol.207, 264-267 (1998), BioSci. Biotecnol. Biochem. Vol.62, No.7, 1461-1464 (1998), Deutsche Lebensmittel-Rundschau, Vol.95, Jahrg., Heft 2, 44-48 (1999), Deutsche Lebensmittel-Rundschau, Vol.95, Jahrg., Heft 2, 48-51 (1999), Deutsche Lebensmittel-Rundschau, Vol.95, Jahrg., Heft 2, 52-56 (1999), Deutsche Lebensmittel-Rundschau, Vol.95, Jahrg., Heft 7, 275-278 (1999), Journal of AOAC International Vol.82, No.4, 923-928 (1999), GIT Labor-Fachzetschrift, 2/99, 156-160 (1999), Bio Industry Vol.16, No.4, 17-21 (1999), Eur. Food Res. Technol. Vol.209, 77-82 (1999), Analytica Chimica Acta Vol.393, 177-179 (1999), Food Control Vol.10, 339-349 (1999), Journal of Agricultural and Food Chemistry, Vol.47, No.12, 5038-5043 (1999), Journal of Food Hygienic Society of Japan, Vol.41, No.2, 137-143 (2000)).
There are also several reports for known techniques that can provide the information of the content ratio of each genetic recombinant contained in the sample such as the reports for techniques for quantitatively determining the recombinant DNA sequences form soybean and maize.
For example, Deutsche Lebensmittel-Rundschau, Vol.95, Jahrg., Heft 2, 57-59 (1999) and Eur. Food. Res. Technol, Vol.209, 83-87 (1999) reports the quantitative detecting techniques for soybean using competitive PCR. These techniques, however, were limited to only a single line of genetic recombinant soybean. Moreover, the accuracy of quantification was relatively low due to the use of competitive PCR and, the purity and yield of the DNA solution extracted from samples affected the result of quantification because they do not use an internal standard.
The quantitative detection technique for genetic recombinant maize using competitive PCR was also reported in Z. Lebensm Unters Forsch A, Vol.207, No.3, 207-213 (1998), but the target of the detection was also limited to only a single line of genetic recombinant maize, which is similar to the above-mentioned case. Furthermore, similarly to the above-described case, the competitive PCR and the lack of internal standard made the reliability of the result of quantification insufficient.
Food Control Vol.10, 353-358 (1999) reports a quantitative detection technique using competitive PCR for genetic recombinant maize and genetic recombinant soybean, but both techniques were limited to only a single line, respectively. Furthermore, the use of competitive PCR and the lack of an internal standard made the reliability of the result of quantification insufficient, as above described.
Quantitative PCR using a fluorescent probe and the like (also referred to as real time PCR or in line PCR etc.) is known as a quantitative analysis which is superior to competitive PCR in the accuracy of quantification. There are several reports regarding the quantitative detection techniques using quantitative PCR for recombinant DNA sequences.
For example, Journal of Agricultural and Food Chemistry, Vol.47, No.12, 5261-5266 (1999) reports that the quantification of recombinant soybean and maize can be performed by quantitative PCR with fluorescent probes. In this reports, the internal standard procedure was employed, which improved the reliability of the quantification result. The target of detection, however, was a single line for genetic recombinant soybean and also a single line for genetic recombinant maize.
Food Control Vol.10, 385-389 (1999) also reports two techniques, the quantitative detection techniques for genetic recombinant soybean using quantitative PCR with fluorescent probes and the quantitative detection techniques for genetic recombinant soybean using competitive PCR. The internal standard procedures were also used in these reports, which improved the reliability of the result of quantification. In contrast to the above-referred reports, the plasmid DNA containing a DNA sequence for internal standard and the plasmid DNA containing the target DNA sequence to be examined were used as a standard molecule. However, since both plasmids were provided separately, there remained the possibility that the manner of diluting the plasmid DNA and the amount of plasmid DNA added into the reaction system affected the result of quantification. Again the target of detection was limited to a single line of genetic recombinant soybean. Chemie in Labor und Biotechnik. Vol.50, Jahrg., Heft 1, 6-8 (1999) is similar to the above-mentioned reports.
Furthermore, since the genetic recombinants that are commercially available as the standard materials are only two lines for maize and one line for soybean, it is very difficult for the analysts to analyze other lines.
A technique for quantifying the content of genetic recombinants was also reported where the protein expressed from a the recombinant DNA sequence is determined by Enzyme Linked Immunosorbent Assay (ELISA), but this technique was also the technique of quantitative quantification for a particular single line.